Photoresponsive prodrug‐dye nanoassembly for in‐situ monitorable cancer therapy

Abstract Photocleavable prodrugs enable controllable drug delivery to target sites modulated by light irradiation. However, the in vivo utility is usually hindered by their insolubility and inefficient delivery. In this study, we report a simple strategy of co‐assembling boron‐dipyrromethene‐chlorambucil prodrug and near‐infrared dye IR783 to fabricate photoresponsive nanoassemblies, which achieved both high prodrug loading capacity (~99%) and efficient light‐triggered prodrug activation. The incorporated IR783 dye not only stabilized the nanoparticles and contributed tumor targeting as usual, but also exhibited degradation after light irradiation and in‐situ monitoring of nanoparticle dissociation by fluorescent imaging. Systemic administration of the nanoparticles and localized light irradiation at tumor sites enabled monitorable and efficient drug release in vivo. Our results demonstrate that such prodrug‐dye co‐assembled nanomedicine is a promising formulation for photoresponsive drug delivery, which would advance the translation of photoresponsive nanomedicines.


| INTRODUCTION
Prodrug strategy has presented potentials in improving the efficacy of chemotherapeutic drugs by conjugating them with functional moieties. 1,2 So far, prodrugs that respond to various stimuli, including pH, 3-6 enzyme, 7,8 ultrasound, 9 heat 10,11 and light, [12][13][14][15] have been developed to reduce the drug toxicity toward normal tissues while retaining their activity at diseased lesions. Light is one of the most convenient and effective triggers that can be spatiotemporally controlled with both high accuracy and low expense. 16 Recently, photoresponsive prodrugs have been developed with tailor-made structures containing photocleavable moieties such as coumarin and boron-dipyrromethene (BODIPY) for photoactivable chemotherapy. [17][18][19] For instance, BODIPYchlorambucil (BC) prodrug, of which the BODIPY group can be efficiently cleaved upon light irradiation, was developed and achieved effective antitumor effect with the utilization of laser light. 20 However, due to the hydrophobicity of most photocleavable groups, water solubility of the reported photoresponsive prodrugs is generally poor, which hindered their utility in vivo. 21,22 Kaiqi Long and Yifan Wang contributed equally to this work.
To solve the problem, prodrug-based nanoparticles, which exhibits good water dispersibility, long-term stability and long circulation time, have been developed. [23][24][25] Several strategies are promoted to fabricate prodrug-based nanoparticles, such as enclosing prodrugs into nanoparticles or adjusting prodrugs with a proper hydrophilic-tohydrophobic ratio for self-assembly. 26,27 For example, hydrophobic photoresponsive prodrugs can be adjusted to amphiphilic molecules by modifying their photocleavable group to a hydrophilic one, which is conducive to molecular self-assembly and the formation of prodrugbased nanoparticles. 18,28 Moreover, co-assembly is also a potent method to fabricate nanomedicines. 29,30 Different molecules can selfassemble into particles through various intermolecular driving forces, such as hydrophobic interaction, electrostatic interaction, etc. The resulted nanoassemblies present serval advantages like facile preparation, safe metabolism, minimal carrier toxicity and thus, convenient co-delivery of different therapeutic agents.
Here, we design and fabricate a photoresponsive prodrug-dye nanoassembly for cancer therapy. IR783, a commercially available dye, has been reported to serve as the stabilizer while forming nanoassemblies with hydrophobic drugs. [31][32][33] In this study, IR783 coassembles with photocleavable BC prodrug into prodrug-dye nanoparticles (IR783/BC NPs; Figure 1a). IR783/BC NPs present great potential for light-controllable drug delivery based on the photocleavage reaction of BC upon light irradiation, followed by the disassembly of the nanoparticles and the release of free chlorambucil (Cb) drug. Interestingly, IR783 in the nanosystem displays multiple functions by serving as (1) a stabilizer that can be degraded after light irradiation; (2) a targeting moiety that enhances the caveolin-1 (CAV-1) mediated transcytosis in tumors; (3) a fluorescent imaging agent for in-situ monitoring of the disassembly of nanoparticles. Thus, IR783/ BC NPs can achieve light-controllable, tumor-targeting and in-situ monitorable cancer therapy (Figure 1b). The efficacy of our system was verified both in vitro and in vivo and the drug release process can be monitored by a in vivo imaging system. The strategy of integrating IR783 and photocleavable prodrug into a multifunctional nanomedicine provides a novel insight for diagnosis and remotely controllable therapy of cancers.

| Preparation and characterization of prodrugdye nanoparticles
The synthesis of photoresponsive BC prodrug followed the published method. 34 As illustrated in Figure 2a, the BC prodrug could F I G U R E 1 Schematic illustration of photoresponsive IR783/BC NP and its therapeutic effect upon light irradiation. (a) Self-assembly of IR783/BC NP and its dissociation upon light irradiation. (b) Cellular uptake of IR783/BC NP by a HCT116 cell and light-triggered drug release for cancer therapy co-assemble with IR783 when injecting the stock solution of BC dropwise in the aqueous solution of IR783 by flash nanoprecipitation method. The excessive IR783 in the solution was removed by centrifugation, and IR783/BC NPs were finally obtained as red-purple dispersion after resuspension in phosphate buffer saline (PBS) (Figure 2b). We first optimized the feeding ratio of IR783 to BC prodrug by adjusting the concentrations of IR783 solution. When the concentration of IR783 solution was 400 μg/ mL, the obtained nanoparticles showed both smallest diameter and lowest polydispersity index (PDI; Figure S1), indicating an optimized mass ratio. Dynamic light scattering (DLS) detected IR783/ BC NPs as nanoassemblies with a hydrodynamic diameter of 87.22 nm and a PDI of 0.089 (Figure 2c). The IR783/BC NPs were negatively charged at À29.70 mV, which attributed to the negatively charged sulfonate groups of IR783 ( Figure 2d). Besides, IR783 is important for assembling nanoparticles. BC prodrug alone, which is highly hydrophobic and water-insoluble, could not form well-dispersed nanoparticles in the aqueous solution while using the same preparation method as IR783/BC NPs ( Figure S2). We then quantified the content of IR783 and BC prodrug in the nanoparticles by high-performance liquid chromatography (HPLC) and calculated the loading capacity and encapsulation efficiency of the two components. As shown in Table S1, we found that both the loading capacity and encapsulation efficiency of BC prodrug were remarkably high, reaching 98.85% and 84.37%, respectively, which demonstrated that BC prodrug was the main component that    Figure S7). In Figure 4a, the peak at 540 nm only displayed slight decrease, since the mixture of BODIPY and Cb has similar absorption with BC, which leads to no significant change of the UV-Vis spectra. Additionally, IR783/BC NPs showed broad NIR fluorescent emission in the region of 780-950 nm that belongs to the emission profile of IR783 dye. 39 The NIR emission displayed an "ON-to-OFF" pattern while applying 530 nm light irradiation on IR783/BC NPs, which also indicated the degradation of IR783 ( Figure 4b). Notably, we did not see similar phenomena while free IR783 was irradiated (Figure 4c). Thus, the photoactivation of BC that   Figure S9). Moreover, IR783 was reported as a tumor-targeting cyanine dye. 45 Based on previous literatures, the nanoparticles coated by indocyanines with sulfonate groups presented caveolae-targeting effect, which was confirmed by the result that both caveolin inhibition and CAV-1 knockout can attenuate the uptake of IR783-incorparated nanoparticles. 31 Similarly, the caveolae-targeting effect of IR783 is also attributed to the efficient cellular uptake of IR783/BC NPs in our study. It should be noted that different cellular uptake in non-cancerous cells, including human these results demonstrate that CAV-1 expression is related to the cellular uptake of IR783/BC NPs.
Furthermore, some internalization pathway inhibitors, including chlorpromazine (clathrin-mediated endocytosis inhibitor), genistein (caveolae-mediated endocytosis inhibitor) and m-β-cyclodextrin (mβ-CD) (lipid rafts-mediated endocytosis inhibitor), and lowtemperature (4 C) incubation were used to analyze the internalization mechanism of IR783/BC NPs. Both genistein and 4 C incubation significantly attenuated the cellular uptake of the nanoparticles, indicating the internalization process dominated by ATP-dependent and caveolae-mediated endocytosis ( Figure S11). Besides, after the internalization, the red signal of IR783 was co-localized with the Lysotracker green signal. The fluorescent signal can stay inside lysosomes for at least 24 h ( Figure S12). After light irradiation, the IR783 signal decreased, indicating that light irradiation can trigger the degradation of IR783, which was consistent with the result shown in To further investigate the therapeutic effect of the nanoparticles, live-dead staining analysis was conducted by Calcein AM/PI costaining. As shown in Figure 5c, the dead cells presenting red fluorescence were observed both in the irradiated BC-treated group and IR783/BC NPs-treated group while other groups without light irradiation did not cause significant cell death, which coincided well with the result of the cytotoxicity study. Additionally, apoptosis study of HCT116 cells treated with free Cb or IR783/BC NPs was conducted by Annexin-V FITC/PI assay to investigate the anticancer activity ( Figure S14). Cell apoptosis rate was significantly elevated after treating with IR783/BC NPs and light irradiation. At the concentration of 20 μM, the nanoparticles plus light irradiation induced 77.13% of cell apoptosis, which was mainly dominated by the late apoptosis (70.45%). In comparison, free Cb at the same concentration only caused about 5% apoptosis no matter whether the light irradiation was applied or not, since the free Cb cannot enter the cells as efficient as the nanoparticles. In all, these results confirmed the effective cytotoxicity and apoptosis-inducing effect of IR783/BC NPs with light irradiation.

| IR783/BC NPs generated ROS upon light irradiation
Interestingly, compared with free Cb, the BC prodrug irradiated with 530 nm light showed higher cytotoxicity. One of the reasons should be that the iodide-containing BODIPY group of BC prodrug exhibits high inter-system crossing efficiency and enhances singlet oxygen   Figure S18). Compared to free IR783, the nanoparticles exhibited much better tumor retention capability. Besides, obviously preferential accumulation of the nanoparticles was also observed in the liver. It should be noted that we can selectively activate the nanoparticles in the tumors by light while those nanoparticles in the liver will not be activated by light, which may reduce side effects.   efficacy of free Cb was undermined in vivo, which may be due to its hydrolysis and fast clearance in blood. 46 The nanoparticles without light irradiation also showed poor efficacy, indicating the therapeutic effect of IR783/BC NPs would only be activated upon light irradiation. After 14-day monitoring, the mice were sacrificed, and the tumors were resected for ex vivo characterizations. The tumor size and weight of the group treated with IR783/BC NPs plus light irradiation were significantly lower than other groups, which further supported the above results (Figure 7d,e). Additionally, no remarkable body weight loss was observed during the treatments with these formulations, suggesting good biocompatibility of the treatments (Figure7f). Moreover, the cell proliferation and apoptosis in tumors were analyzed by hematoxylin and eosin (H&E) staining and immunohistochemistry staining, respectively. The results shown in Figure 8 revealed that IR783/BC NPs with light irradiation effectively inhibited proliferation and induced apoptosis in the tumor tissues. As expected, immunohistochemistry staining of CAV-1 also confirmed its high expression in all the tumors. As compared to the normal tissues (heart, liver, spleen, lung, and kidney), a larger area in the tumor tissue displayed CAV-1 positive (brown areas in Figure S19). Moreover, based on the H&E staining results (Figure S20), the main organs exhibited no apparent necrosis or histological change after the treatments,

| IR783/BC NPs plus light irradiation efficiently inhibited HCT116 tumor growth in vivo
suggesting minimal systemic toxicity. The secretion of some of the liver enzymes such as alanine aminotransferase and aspartate aminotransferase in the serum of the mice after different treatments was investigated and no obvious differences between all the groups were found ( Figure S21). All these in vivo data suggested that the IR783/ BC NPs could serve as a promising nanomedicine against tumors with light-controllable activity, resulting in enhanced antitumor efficacy and biosafety.

| CONCLUSIONS
Herein, we designed a simple yet novel strategy to fabricate photoresponsive nanomedicine by co-assembly of photocleavable prodrug and IR783 dye. Interestingly, the incorporation of IR783 as the stabilizer enabled both high loading capacity of BC prodrug Light-responsive drug delivery systems have been shown to be useful for cancer treatment by applying light to trigger drug release and accumulation. The wavelength of light used for photoresponsive drug delivery is of great importance. 12 The most frequently used light for the activation of the systems is UV (<400 nm) light, of which the tissue penetration is limited. 52,53 The longer wavelength light would allow deeper tissue penetration. 53,54 In this study, green light was used, which was reported to trigger the phototargeting of nanoparticles in a subcutaneous tumor model 40 and trigger drug release from nanoparticles in the eye. 55 The results of in-situ fluorescence monitoring and tumor growth inhibition indicated that the 530 nm green light can reach the subcutaneous HCT116 tumors for light-triggered cancer therapy. Therefore, such nanomedicine may be applied for drug delivery to superficial or easily accessible tissues, like skin or eyes. For diseased lesions deep in the body, light delivery by optical fibers may be an option. 56,57 IR783 presented versatile functions by serving as a stabilizer, tumor-targeting moiety, and imaging agent in our system. Though IR783 was widely used for imaging in clinic, 58 its application in drug delivery has just taken its first step. Some nanosystems that involved IR783 as a stabilizer have been reported recently. 31,42 Nevertheless, for the first time, this work demonstrated IR783 as a "three-birdswith-one-stone" agent in a simple prodrug-dye co-assembled nanoparticle, especially as a reporter for in-situ monitoring of nanoparticle dissociation and drug release. IR783 exhibits such functions because of its integration in the photoresponsive system, for example, the photoresponsive prodrug-dye nanoparticles in this study.
In summary, a simple and reliable co-assembly strategy for developing photocleavable prodrug-dye co-assembled nanomedicine was reported. It is a promising platform for precisely and remotely controllable cancer therapy. The study may open a new path for fabricating multifunctional photoresponsive drug delivery systems with ultrahigh loading capacity in a simple, reliable, and clinically transferable way.
Other photoresponsive prodrugs, especially long-wavelength lightexcitable prodrugs, are worthy of being explored to form similar nanomedicines for specific disease treatment.

| Materials and instruments
Indocyanine IR783 dye was purchased from Tokyo Chemical Industry (TCI) Co., Ltd. Chlorambucil (Cb) was purchased from J&K Scientific.   The flow rate of the mobile phase was 1.5 mL/min. Experiments under each set of conditions were repeated for at least three times.

| Spectrum study of IR783 under light irradiation
The degradation of IR783 was demonstrated by spectroscopy. The

| Singlet oxygen detection
Singlet oxygen generation was measured both in solutions and cells using SOSG and DCFH-DA as indicator, respectively. Briefly, free IR783, free BC, and IR783/BC NPs (at the equivalent concentration of BC at 1 μM) was mixed with SOSG probe (5 μM) in PBS.
The solutions were exposed to light irradiation and the fluorescence intensity was detected at predetermined time inter- At 24 h, the mice were euthanized and tumors and major organs (heart, liver, spleen, lung, and kidney) were excised for ex vivo imaging.
4.14 | In-situ monitoring of light-triggered therapy

| Statistical analysis
All experiments were conducted three times or more independently (n ≥ 3). Data were presented as the mean ± standard deviation (SD).
The one-way ANOVA-LSD and Independent Samples t-test were adopted to determine the statistical significance of differences by Graphpad Prism 8.0 software.

CONFLICT OF INTERESTS
A US provisional patent application was filed with No. 63/262,789.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.